Single nucleotide changes in human genes and surrounding DNA may cause genetic disorders and are currently believed to reveal the cause of individual susceptibility to different diseases. The most common type of genetic diversity is single nucleotide polymorphism (SNP). Therefore, accurate and sensitive analysis of SNPs may play a central role in DNA diagnostics. Analysis of SNPs is useful in applications including mapping, linkage studies and pharmacogenoics.
A number of new methods are now available for rapid automated scoring of SNPs (A. Ahmadian and J. Lundeberg, 2002, Biotechniques, 1122-1137). Many methods originate from hybridization techniques to discriminate between allelic variants. The use of microarrays with oligonucleotide reagents immobilized on small surfaces in miniaturized assays is a frequently proposed approach for large-scale mutation analysis and high-throughput genotyping of SNPs (D. Wang et al, 1998, Science, 1077-1082). Microarray hybridization of PCR fragments to allele-specific oligonucleotide probes (ASO) relies on the thermal stability of the PCR fragment and the short probe and has been used in large-scale SNP genotyping. However, the limitation of this technique lies in the fact that there are extremely small differences in the duplex stability between a perfect match and a mismatch at one base and therefore, for accurate analysis this approach requires redundant probes. To enhance the discrimination power of microarray based mutation/polymorphism analysis, enzymatic reactions have been performed on the oligonucleotide arrays (J. Hirschhorn et al, 2000 PNAS, 12164-12169 and T. Pastinen et al, 2000, Genome Research, 1031-1042).
Other technologies which have been shown to be useful for SNP genotyping are minisequencing (Pastinen et al. (1997) Genome Res. 7: 606), mass spectrometry (Laken et al. (1998) Nat. Biotechnol. 16: 1352) and Pyrosequencing (Ahmadian et al. (2000) Anal. Biochem. 280: 103), the latter relying on incorporation of nucleotides by DNA polymerase with an enzymatic cascade converting the released pyrophosphate (PPi) into detectable light.
The use of pairs of allele-specific primers with alternative bases at the 3′ end has been used to identify single base variations. Higgins et al. (1997) Biotechniques 23: 710; Newton et al. (1989) Lancet 2: 1481; and Goergen et al. (1994) J. Med. Virol. 43: 97. This method exploits the difference in primer extension efficiency catalysed by a DNA polymerase of a matched over a mismatched primer 3′-end. Generally, a sample is divided into two extension reaction mixtures that contain the same reagents except for the primers, which differ at the 3′-end. The alternating primer is designed to match one allele perfectly but mismatch the other allele at the 3′-end. Because the polymerase differs in extension efficiency for matched versus mismatched 3′-ends, the allele-specific extension reaction thus provides information on the presence or absence of one allele.
Allele-specific extension has traditionally been applied in the context of genetic variation such as allelism where a given DNA sequence (e.g., a gene) occupying a particular locus on a chromosome or linkage structure may occur in two or more alternate and different forms (typically biallelic variation where two alternate alleles exist, but occasionally there may be three or four or more (where more than single base changes are concerned) different allelic forms). Similarly, the same principle may be used to distinguish different mutations or base changes which may occur at a given locus or target site (i.e. “mutation-specific” primers, by analogy).
It will be appreciated from this, therefore, that the basic principle of “allele-specific” discrimination by “allele-specific” primer extension can be applied to any situation where genetic variation can occur at a given locus or target site, and that such variants or differences can be discriminated by using primers designed to be specific for the particular variations (variants) or base differences concerned. Thus, for example, in the case of genotyping applications, where it may be desired to detect or determine what base(s) is (are) present at a particular location(s), it will be seen that not all organisms (e.g. viruses) may exhibit allelism (i.e. carry two or more alleles), but different forms or strains (types) thereof may be distinguished (or “typed”) using this general principle, namely the use of different specific primers, each specific for a particular “type”, or variant base which may occur (i.e. “type”- or “variant”-specific primers). “Allele-specific” extension may thus be recognised as extending to any situation where a polymerase enzyme, for example DNA or RNA polymerase or reverse transcriptase, is used to extend a primer which is matched, but not a primer which is mismatched, in the context of any given base change or genetic variation, where a primer may “match” one of the variants which may occur, and hence “mismatch” the other.
Allele-specific primer extension takes advantage of the discrimination properties of a polymerase in extension of a 3′-end matched primer. This method has been employed previously to identify single base variations (C. Newton et al, 1989, Nucleic Acids Research, 2503-2516) but it is generally acknowledged that certain mismatches, such as G:T or C:A are poorly discriminated by the employed polymerase enzyme. This poor discrimination property of the polymerase has consistently been observed in applications of this technique (S. Ayyadevara et al, 2000, Analytical Biochemistry, 11-18, J. Day et al, 1999, Nucleic Acids Research, 1810-1818 and S. Kwok et al, 1990, Nucleic Acids Research, 999-1005). In these cases, the polymerase extends the mismatched primer-templates in the presence of nucleotides.
The applicants have previously shown that the discriminatory ability of the polymerase enzyme in the context of “allele-specific” extension assays depends upon relative reaction kinetics. Extension of mismatched primer-template configurations occurs with slower reaction kinetics in comparison to the faster extension of matched primer-template configurations. The kinetic difference is usually not distinguishable in end-point analysis, such as in allele-specific PCR, because extension of a single mismatched substrate in the first cycle will lead to perfectly matched primer-templates in subsequent cycles, yielding comparable amounts of end products for both matched and mismatched configurations.
The applicants have further previously demonstrated that the use of a nucleotide-degrading enzyme (particularly apyrase) during the assay improves discrimination in allele-specific extension assays by exploiting this difference in the relative kinetics (A. Ahmadian et al, 2001, Nucleic Acids Research, Volume 29, No. 24 e121 and D. O'Meara et al, 2002, Nucleic Acids Research, Volume 30 No. 15 e75 and WO 02/068684 of Pyrosequencing AB. In apyrase-mediated allele-specific extension (AMASE), when the reaction kinetics are fast (i.e. with a matched primer) the primer-template is extended by the polymerase before the apyrase (or other nucleotide-degrading enzyme(s)) degrades the nucleotides. However, when the reaction kinetics are slow, due to the mismatched primer, apyrase degrades the nucleotides before they can be incorporated and prevents or hinders extension of the mismatched primer-template.
Although the use of apyrase (or other nucleotide-degrading enzyme) has circumvented some of the main difficulties of allele-specific primer extension assays, there are some disadvantages related to this technique. Primarily, apyrase degrades the nucleoside triphosphates to nucleoside diphosphates and there can be a problem with contamination with kinases in the reaction mixture that are able to convert any nucleoside diphosphates back to nucleoside triphosphates. These nucleoside triphosphates may then be used by the polymerase to extend a mismatched primer-template. One solution to this problem may be to use an additional enzyme that is able to degrade the nucleoside diphosphates to nucleoside monophosphates. This, however, has the disadvantage of proliferating reagents. Secondly, there is evidence available that apyrase binds to DNA templates and primers (M. Ronaghi, 2000, Analytical Biochemistry, 282-288). The binding of apyrase to DNA makes the enzyme unavailable in the reaction mixture, particularly when the quantity of DNA to be analysed is high. One solution to this problem is to use additional protein in the assay. This protein may be single-stranded DNA binding protein (SSB). SSB binds to single-stranded DNA and releases apyrase into the reaction mixture. Again, however, this entails the use of further reagents. Thirdly, apyrase is not a thermostable enzyme and is inactive at higher temperatures, which is a considerable disadvantage when more stringent temperature conditions are required. It is well-known in molecular biotechnology that at lower temperatures the DNA forms different secondary structure, including loop formation. In addition to DNA loops, lower temperature is the major cause of non-specific hybridization. These aspects can cause problems in the AMASE assay which requires lower temperatures to allow the apyrase to perform the degradation of nucleotide triphosphates. Formation of DNA loops on mismatched oligonucleotide primers gives rise to self-extension of the primers leading to incorrect results. Non-specific hybridization is a major problem especially in multiplex genotyping assays (e.g. where multiple primers are present), giving rise to false signals. This problem can in most cases be circumvented by using higher and more stringent temperature conditions. However, the stringent temperature conditions cannot be achieved by the AMASE assay without significant loss of apyrase activity.
There is thus a continuing need in the art for improved methods of allele-specific extension analysis and the present invention addresses this need.
The method of the present invention solves the deficiencies of the prior art methods by providing a method of allele-specific or type-specific extension that allows more accurate discrimination between matched and mismatched configurations. The present methods are useful for any type of genetic analysis based on detecting or identifying particular or specific base changes, for example high throughput SNP analysis and genotyping assays. The present methods may be used also for detecting or screening for mutations (such as substitutions, insertions and deletions) and genomic variations, and particularly single nucleotide polymorphisms (SNPs), and may be used with single stranded or double stranded DNA targets.